COPII proteins play a critical role in the early secretory pathway by transporting protein and lipid cargo out of the endoplasmic reticulum (ER). The COPII proteins consist of Sar1 (a GTPase), Sec23/24 (cargo selection and GAP activity), and Sec13/31 (promotes coat assembly). Together, these form a coat that recruits and concentrates cargo and gradually deforms the ER membrane into a vesicle. Recently, it was found that Sec13/31 self-assembles into a unique cuboctahedron cage-like structure. These structures were solved with cryo-electron microscopy (cryoEM) and single particle reconstruction and yielded insights into some of the mechanisms by which COPII coated vesicles are assembled. These initial studies were bolstered with a new structure of a COPII coat formed from the self-assembly of Sec13/31 with Sec23/24. Together, these two structures form a foundation for dissecting the mechanisms by which the COPII proteins perform their functions in the cell. The current proposal seeks to address questions about COPII structure and assembly through four specific aims. Aim 1 proposes to determine the structures of individual COPII coats and COPII coated vesicles. These structures will shed light on the ways that cargo interacts with Sec23/24 and Sec23/24 interacts with Sec13/31, and thus contribute to a picture of the mechanisms by which cargo directs the assembly of vesicles of the proper size. In aim 2, it is proposed to determine the structure of the COPII coat in complex with Sar1, and these studies will reveal at a molecular level how Sar1 is involved in initiating the formation of the COPII coat. The molecular mechanisms of tethering are explored in aim 3, where it is proposed to determine the structure of the COPII coat in complex with the TRAPPI tether protein Bet3. Finally, aim 4 proposes to develop assays for poisoning COPII cage assembly at various intermediate stages and to determine the structures of the intermediates. Together, these studies will drive the vesicle transport field by furthering our understanding of COPII structures and how they are assembled in the cell. This, in turn, will aid in the understanding of the role COPII proteins play in diseases like chylomicron retention disease and cranio-lenticulo-sutural dysplasia, which result from mutations in Sar1 and Sec23/24 respectively, and diseases that manifest as transport defects such as cystic fibrosis.